The goal of this research proposal is to regenerate the intervertebral disc (IVD) by creating scaffolds which mimic the IVD microstructure and mechanics to engineer an in vivo IVD regeneration cascade. Disc degeneration is highly prevalent in the aging population, resulting in a deterioration of the complex IVD structure and ultimately sacrificing the ability of the IVD to function properly. IVD pathologies include age related degeneration as well as noticeable pain and a loss in patient mobility. Many factors have been implicated in disc degeneration including aging, excessive mechanical loading, and a decrease in IVD cell nutrition, as the IVD is the largest avascular tissue in the body. Current solutions to treat IVD degeneration, which include discectomy, spinal fusion, and the use of artificial disc replacements, do nothing to support natural tissue regeneration and focus only on the symptoms of disc degeneration. Discectomy promotes further compression of the spinal nerves and an increase in patient pain, while current implants exhibit a large compliance mismatch from the native tissue, resulting in further IVD degeneration at adjacent spinal levels. Therefore, our hypothesis is that the creation of a 3D scaffold that precisely mimics the complex microstructure and biomechanics of the native IVD may be suitable to promote IVD regeneration in vivo when combined with appropriate signaling molecules to recruit and proliferate endogenous stem cells while promoting chondrogenic differentiation, ultimately leading to structural and functional tissue recovery. Aim 1: In vitro assessment of the ability to control differentiation of mesenchymal stem cells (MSCs) into IVD cell phenotypes. The working hypothesis is that MSC differentiation can be regionally controlled using different combinations of biomolecules to produce specific cell lineages within the IVD. We have already discovered candidate signaling molecules to encourage stem cell migration and proliferation within the scaffold. Therefore, optimized differentiation cocktails will be created for each IVD region to support gene expression similar to native IVD tissue. Aim 2: Assess in vivo growth of IVD tissue by recruiting endogenous stem cells and controlling proliferation and IVD phenotypic differentiation. The working hypothesis is that with the proper combination of signaling molecules, the biphasic scaffolds will recruit endogenous stem cells and encourage proliferation, chondrogenically differentiate, and synthesize ECM similar to that of native IVD tissue. Scaffolds will first be created with immobilized signaling molecules followed by implantation and histological evaluation in subcutaneous rodent models.